A Comparative Genomic Approach to Determine the Virulence Factors and Horizontal Gene Transfer Events of Clinical Acanthamoeba Isolates

ABSTRACT Acanthamoeba species are among the most ubiquitous protists that are widespread in soil and water and act as both a replicative niche and vectors for dispersal. They are the most important human intracellular pathogens, causing Acanthamoeba keratitis (AK) and severely damaging the human cornea. The sympatric lifestyle within the host and amoeba-resisting microorganisms (ARMs) promotes horizontal gene transfer (HGT). However, the genomic diversity of only A. castellanii and A. polyphaga has been widely studied, and the pathogenic mechanisms remain unknown. Thus, we examined 7 clinically pathogenic strains by comparative genomic, phylogenetic, and rhizome gene mosaicism analyses to explore amoeba–symbiont interactions that possibly contribute to pathogenesis. Genetic characterization and phylogenetic analysis showed differences in functional characteristics between the “open” state of T3 and T4 isolates, which may contribute to the differences in virulence and pathogenicity. Through comparative genomic analysis, we identified potential genes related to virulence, such as metalloprotease, laminin-binding protein, and HSP, that were specific to the genus Acanthamoeba. Then, analysis of putative sequence trafficking between Acanthamoeba and Pandoraviruses or Acanthamoeba castellanii medusaviruses provided the best hits with viral genes; among bacteria, Pseudomonas had the most significant numbers. The most parsimonious evolutionary scenarios were between Acanthamoeba and endosymbionts; nevertheless, in most cases, the scenarios are more complex. In addition, the differences in exchanged genes were limited to the same family. In brief, this study provided extensive data to suggest the existence of HGT between Acanthamoeba and ARMs, explaining the occurrence of diseases and challenging Darwin’s concept of eukaryotic evolution. IMPORTANCE Acanthamoeba has the ability to cause serious blinding keratitis. Although the prevalence of this phenomenon has increased in recent years, our knowledge of the underlying opportunistic pathogenic mechanism maybe remains incomplete. In this study, we highlighted the importance of Pseudomonas in the pathogenesis pathway using comprehensive a whole genomics approach of clinical isolates. The horizontal gene transfer events help to explain how endosymbionts contribute Acanthamoeba to act as an opportunistic pathogen. Our study opens up several potential avenues for future research on the differences in pathogenicity and interactions among clinical strains.

1. Line 65-66: To date, 23 genotypes of acanthamoeba are known, i.e., T1-T23. According to a recent study, acanthamoeba has been isolated from public freshwater sources in Thailand and named as A. bangkokensis, and it belongs to the T23 genotype. (doi: 10.1038/s41598-021-96690-0) that need to be updated in the introduction section. 2. Line 82-83 what does mean "selective grazing growth." As per my understanding, it reflects phagocytosis. So my question is phagocytosis in acanthamoeba specific or non-specific? 3. Line 85-86: How can acanthamoeba resist phagocytosis? It is reported that endosymbiont survives intracellularly inside acanthamoeba and resists acanthamoebacidal digestion. However, I can't understand how acanthamoeba can resist phagocytosis. 4. Italic acanthamoeba and all bacteria names throughout the text. 5. Line 121 authors mentioned that they had studied the "prevalence of endosymbionts." However, results are nowhere mentioned and discussed, and seven is a minimal sample size to determine prevalence. 6. In the material and method, it is mentioned that to culture acanthamoeba, non-nutrient agar method was used, i.e., monoxenic culture, which includes cultivation of acanthamoeba in the presence of bacteria such as E.coli. How about axenic culture .i.e. without any bacteria? For all genomic studies, DNA extraction should be done with pure culture. DNA extracted from monoxenic culture probably has E.coli DNA as well. Justify it 7. Line 353: write the full form of ARB and ARVs 8. A horizontal gene transfer study was done with new and publicly available data. Have authors tried to validate the same with in vitro experiments 9. Line 475-477 authors have identified several endosymbionts, such as Chlamydiae, Mycobacterium, Pseudomonas, Legionella, Burkholderia, by genomic study. Have you seen them on the bacterial culture of any other methods to verify your results as well? 10. Add bootstrapping value in the phylogenetic tree.
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The research article by Gu et al. examined seven clinically proven pathogenic strains of acanthamoeba and compared them with those of acanthamoeba and other amoebae from public databases to study genomic, phylogenetic, and identification of virulence genes specific to acanthamoeba. The authors also demonstrated rhizome gene mosaicism analyses to explore amoeba symbiont interactions that possibly contribute to pathogenesis. Though the manuscript is interesting, well-written, and easy to read and understand, I still have some suggestions/ concerns that need to be addressed before this manuscript is published. My suggestions/concerns are listed below: 1. Line 65-66: To date, 23 genotypes of acanthamoeba are known, i.e., T1-T23.
According to a recent study, acanthamoeba has been isolated from public freshwater sources in Thailand and named as A. bangkokensis, and it belongs to the T23 genotype. (doi: 10.1038/s41598-021-96690-0) that need to be updated in the introduction section. 2. Line 82-83 what does mean "selective grazing growth." As per my understanding, it reflects phagocytosis. So my question is phagocytosis in acanthamoeba specific or non-specific? 3. Line 85-86: How can acanthamoeba resist phagocytosis? It is reported that endosymbiont survives intracellularly inside acanthamoeba and resists acanthamoebacidal digestion. However, I can't understand how acanthamoeba can resist phagocytosis. 4. Italic acanthamoeba and all bacteria names throughout the text. 5. Line 121 authors mentioned that they had studied the "prevalence of endosymbionts." However, results are nowhere mentioned and discussed, and seven is a minimal sample size to determine prevalence. 6. In the material and method, it is mentioned that to culture acanthamoeba, non-nutrient agar method was used, i.e., monoxenic culture, which includes cultivation of acanthamoeba in the presence of bacteria such as E.coli. How about axenic culture .i.e. without any bacteria? For all genomic studies, DNA extraction should be done with pure culture. DNA extracted from monoxenic culture probably has E.coli DNA as well. Justify it 7. Line 353: write the full form of ARB and ARVs 8. A horizontal gene transfer study was done with new and publicly available data. Have authors tried to validate the same with in vitro experiments 9. Line 475-477 authors have identified several endosymbionts, such as Chlamydiae, Mycobacterium, Pseudomonas, Legionella, Burkholderia, by genomic study. Have you seen them on the bacterial culture of any other methods to verify your results as well? 10. Add bootstrapping value in the phylogenetic tree.

Reviewer #1
The research article by Gu et al. examined seven clinically proven pathogenic strains of acanthamoeba and compared them with those of acanthamoeba and other amoebae from public databases to study genomic, phylogenetic, and identification of virulence genes specific to acanthamoeba. The authors also demonstrated rhizome gene mosaicism analyses to explore amoeba symbiont interactions that possibly contribute to pathogenesis. Though the manuscript is interesting, well-written, and easy to read and understand, I still have some suggestions/ concerns that need to be addressed before this manuscript is published.

Response:
We sincerely appreciate all your positive comments. The suggestions/concerns were addressed in detail as below. Response: Thank you so much for pointing this out. We have updated the introduction section accordingly. Please refer to correspondingly text on Line 65-66 in our revised manuscript.

Line 82-83 what does mean "selective grazing growth." As per my understanding, it reflects phagocytosis. So my question is phagocytosis in acanthamoeba specific or non-specific?
Response: Thank you so much for your question. We should have described it much clearer.
We have briefly mentioned in our manuscript that Acanthamoeba feed on bacteria, fungi, yeasts, algae during the trophozoite stage. Protozoan grazing is believed to be the major trophic pathway whereby the biomass production and amoebae is indicated to a role as bacterial regulators which help to drive bacterial virulence and may contribute to the spread of antibiotic resistance. The amoebae can exert a pressure of selection on the microorganisms that live in their environment and select the microorganisms able to live inside cells in any situations. In fact, any object larger than 0.5 μm can be engulfed, including latex beads. Therefore, the phagocytosis in acanthamoeba is non-specific which is not needed for recognizing specific markers on microorganisms to ingest them. And the phagocytosis of microorganisms follows the endocytic pathway to be degraded in acidic phagolysosomes by a number of hydrolases. In our revised manuscript, we have modified the corresponding part to make it more understandable.

Justify it
Response: We sincerely appreciate your valuable comments. We understand your concern and your suggestion really means a lot to us. We have briefly described the cultivation method in the method part that Acanthamoeba are grown on agar-agar plates seeded with Escherichia coli (ATCC25922) and Page's modified Neff's amoeba saline. To further identify virulence factors and endosymbionts in Acanthamoeba, we removed scaffolds that originated from E.coli. On the one hand, E.coli becomes prey for the the amoebae among all relationships while we aim to find pathogenic endosymbionts in Acanthamoeba. On the other hand, the pathogenic effect of E.coli remains largely unrevealed, and whether it has a pathogenic effect remains to be explored. Axenic culture is a very good option to study Acanthamoeba without interference, and we have started to adopte your suggestion and apply it to the next research.
Besides, the role of E.coli in AK will also become our future research direction.

Line 353: write the full form of ARB and ARVs
Response: We thank the reviewer for point this out. We feel really sorry for our carelessness.
We have rectified the full form of ARB and ARVs in our revised manuscript.

A horizontal gene transfer study was done with new and publicly available data. Have authors tried to validate the same with in vitro experiments.
Response: Thank you for your valuable comment. We're sorry that we cannot timely present the vitro experiments based on current research findings. At present, we have started research on experiments verification, and in the next research, we will explore these interesting results with more evidence.

Response:
We sincerely appreciate all your positive comments. We understand your concern.
Please also find the following changes we have made, which were detailed below.

Rows 71-74, split sentence
Response:Thanks for your help. Based on your comments, we have rearranged and rewrote sentences on page 4 in the revised manuscript to make it more comprehensible.

Row 79 Unsure what "shortening of clinical course" means
Response: We thank the reviewer for point this out. We should have described it much clearer. Acanthamoeba keratitis is often misdiagnosed and treated as herpetic, bacterial, or mycotic keratitis, as many signs and symptoms may look similar to other kinds of keratitis. It is challenging for an ophthalmologist to find the right diagnosis. Therefore, diagnosis is often delayed and ophthalmologists tend to observe a heterogeneous and protracted clinical course.
We have corrected the formulation on page 4 in the revised manuscript. Response: Thank you for this important point. We understand your concern and your suggestion really means a lot to us. As a matter of fact, there exists no readymade virulence gene bank for Acanthamoeba in the Virulence Factor Database (VFDB). There are still many limitations in the mining of virulence genes and the pathogenesis mechanism of AK remains unknown. In our study, our choose of comparative genomic analysis to find virulence genes lay the groundwork for further digging into pathogenesis in the future. We will gradually complete the Acanthamoeba virulence gene pool in the next study. Response: Thank you for this important point. We have identified orthologous genes using Proteinortho with some controls. To minimize highly critical genes loss, we lowered the control limits for coverage and identity and still did not find the mannose binding protein gene. The results may indicated that genomic insufficiency or similar infection pathways in different Acanthamoeba, which were needed to be clarified in the future. We have also modified the discussion of this conclusion, please refer to part accordingly in our revised version.

Response:
We feel great thanks for your professional review work on our article. The stable